The invention concerns scaffold matrixes for supporting three-dimensional tissues and systems for maintaining three-dimensional viable tissues.
The following publications are believed to be relevant as background of the invention.
WO 98/22573
U.S. Pat. No. 4,880,429
U.S. Pat. No. 4,108,438
U.S. Pat. No. 5,843,182.
Cartilage is a specialized form of connective tissue composed of cells and matrix. The cartilage cells synthesize matrix and become encased in cavities (lacunae) within it. The matrix is composed of fibers embedded in ground substance and endows cartilage with its specialized physico-chemical properties.
Trauma, single or repetitive, is the most known cause of damage and degeneration of articular cartilage, that leads to pain, chronic disability and ultimately to joint failure. The current options for treatment provide temporary improvement of symptoms and function, however, there is no full restoration of joint performance. Prosthetic joint replacement is currently the ultimate and the most commonly employed treatment. Modem biological grafting is the other alternative for resurfacing the damaged joint, but is still imperfect.
A large number of candidate grafts have been studied for enhancing the repair of cartilage defects which include: (i) Osteochondral graft (autografts or allografts); (ii) Intact cartilage grafts; (iii) Growth plate; (iv) Isolated allogeneic chondrocytes; (v) Cultured autologous chondrocytes (dedifferentiated) (vi) Periosteum; (vii) Perichondrium; (viii) Bone marrow mesenchymal derived cells and (ix) Synovial membrane.
Another approach was the attempt to use natural occurring or synthetic biodegradable scaffolds which support three-dimensional growth of cartilage cells. The scaffolds may be impregnated with cells, which together with the scaffold form the graft. Alternatively, the scaffold may initially be devoid of impregnated cell, and endogenous cells from the patient are expected to migrate into the scaffold after its implantation.
Examples of such scaffolds are: (a) Fibrin polymers; (b) Collagent Type I; (c) Natural hyaluronic acid (HA) and chemically modified HA and (d) Synthetic bipolymers either biodegradable or non-biodegradable (e.g. alginic acid) and (e) Polylactic acid, polyglycolic acid. However, none of the above scaffolds can induce generation of hyaline-like cartilage. Fibrin polymers tend to induce dedifferentiation and thus do not permit production of functional tissue. Collagen Type I has no inherent chemotactic ability for chondrocytes, but stimulate proliferation of fibroblast. Thus instead of encouraging migration of chondrocytes the tissue formed in this scaffold tends to be fibrous. Hyaluronic acid can stimulate chondrogenic differentiation, but does not stimulate chondrocytes proliferation. Alginic acid is a foreign cabohydrate and thus might induce an antigenic reaction, and furthermore is not biodegradable. Polyglycolic and polylactic acid scaffolds do not support good hyaline cartilage regeneration due to acidic conditions during degradation.
Damaged or missing hyaline cartilage is frequently repaired by transplantation of homografts. Homografts are immunologically privileged the matrix acts as a barrier that permits only limited diffusion of high-molecular weight substances and contains an anti-angiogenesis factor to prevent invasion of host blood vessels and fibroblasts.
Various culturing systems have been developed for maintaining the viability and growth of tissues in culture. Generally, these are divided into static and perfusion bioreactors. Perfusion bioreactors are reactors which essentially keep constant, growth permissible conditions (such as gas composition, temperature, pH, etc.) in which the growth fluid medium is constantly perfused in and out of the system. Typically, perfusion is carried out by utilizing a constant velocity flow of the medium.
By a first aspect, the present invention concerns a scaffold for use as growth supportive base for cells and tissue explants from three-dimensional tissue, comprising naturally derived connective or skeletal tissue which has been treated for elimination of cellular and cytosolic elements, and which has been modified by cross-linking with an agent selected from the group consisting of: hyaluronic acid, proteoglycans, glycosaminoglycan, chondroitin sulfates, heparan sulfates, heparins and dextran sulfates.
It has been found that such a scaffold has the properties of encouraging cells adherence thereto and enablement of propagation of cells on the one hand, while the cross-linking with the agents specified above gives the scaffold mechanical strength, produces a substance which is less brittle with prolonged degradation time on the other hand. It was further found that the scaffold of the invention supports chondrocyte proliferation at the expense of fibroblasts, resulting in a hyaline-like repair tissue.
The term xe2x80x9cscaffoldxe2x80x9d in the context of the present invention refers to the connective/skeletal tissue which has been treated for elimination of cellular and cytosolic agents and modified by cross-linking as described above, as well as to such a construct containing additional agents such as adhesive molecules or growth factors.
The term xe2x80x9cthree-dimensional tissuexe2x80x9d (3D tissue) refers to any type of tissue which has an orderly three-dimensional structure, i.e., is not naturally present in the body in the form limited to a single layer of cells or lamina, but has a stucture which is spatially ordered. Examples of three-dimensional tissue are: mesenchymal tissue, cartilage and bone tissue, liver tissue, kidney tissue, neuronal tissue, fibrous tissue, dermis tissue etc. Another three-dimensional tissue is the whole embryonal epiphyseal organ derived from embryos at a post limb-bud stage.
The naturally derived connective or skeletal tissue is, in general, tissue that was derived from mesenchymal tissues that express, temporarily or continuously fibroblast growth factor receptor 3 (FGFR3). Examples of such tissue are mainly members of chondrogenic and osteogenic anlagen, as well as the residual mesenchymal stem cell reservoirs found in tissues all along life, ready to carry wound healing, repair and regeneration tasks. Another example of connective or skeletal tissue is epiphyseal tissue.
The tissue should be treated for elimination of cellular and cytosolic elements such as: DNA, RNA, proteins, lipids, proteoglycans and in general most elements of the cells which are immunogenic, as well as treated for removal of calcification-mineralization centers. Methods for elimination of the above cellular and cytosolic elements are in general known in the art.
The naturally derived connective or skeletal tissue treated as described above for elimination of cellular and cytosolic components is preferably further treated for producing porosity by the production of pores in a controlled manner. The treatment may be mechanical, for example, by hammering the tissue on a scraper device.
Alternatively, the treatment for producing porosity may be a chemical extraction process carried out by exposing the tissue, for a controlled amount of time, in a controlled environment to chemical agents capable of degradation of the tissue. In addition or alternatively, the treatment for producing porosity may be by exposing the tissue to enzymatic agents such as proteolytic enzymes, capable of partial degradation of the tissue. Example of such chemical agents which can produce pores in the tissue are guanidium chloride. The pores should have preferably a size of 10-500xcexc, most preferably 20-100xcexc.
The agents either specified in above (i.e. hyaluronic acid, proteoglycans, glycosaminoglycan, chondrotin sulfates, heparan sulfates, heparin and dextran sulfates) or additional agents such as adhesive molecules or growth factor moieties may be linked to the residual scaffold either by sugar cross-linking, (for example using ribose and xylose) or by carbodiimideor 1, 1 carbonyl di-imidazole. Cross-linking with the above agents is generally carried out as known in the art of coupling in organic chemistry.
In accordance with the present invention, it is preferable that the scaffold would also contain adhesive molecules in order to enhance cell adherance to the scaffold. Example of suitable adhesive molecules are the integrins and additional agents such as, laminin, fibronectin, hyaluronic acid, polylysine and lysozyme.
In accordance with the present invention, it is also preferable that the scaffold would contain growth factors, in order to enhance the rate of growth of the cells filling the three-dimensional space of the scaffold. Examples of suitable growth factors are: fibroblast growth factors (FGF""s), TGF""s, BTP""s, IGF""s. The growth factor chosen should depend on the type of tissue used.
As indicated above, the adhesive molecules and growth factor molecules should be made part of the scaffold by cross-linking as explained above.
By one option, it is possible to formulate a prosthesis from the scaffold alone, i.e. of a scaffold devoid of cells. In such a case, the scaffold is formulated to a desired shape and is inserted into the desired location in the body of the individual, for example, a location wherein it is desired to achieve invasion of endogenous mesenchymal cells such as in the knee joint.
The prosthesis is maleable and can be shaped as either a flat sheet of several millimeters in thickness or any other three-dimensional shape adapted to the shape of the lesion. Alternatively, the prosthesis, can, a priori, prior to implantation contain embedded (impregnated) cells or tissue explants to allow their fast anchorage and integration into bone and cartilages.
The cells impregnating the prosthesis should preferably be from an autogeneic source, but can also be of an allogeneic source, as cartilage has a sort of an immunoprivilage.
The scaffold of the invention impregnated with cells may be used, not only for implanting in the body but also for prolonged in vitro growth and differentiation of various three-dimensional tissues kinds such as skin, neuronal, bony, cartilaginous, liver, pancreatic beta cell and almost of any organ or tissue in a bioreactor, while adjusting the proper medium, coctail of growth factors and adhesive molecules.
By another aspect the present invention concerns a system for maintaining viable three-dimensional tissue. In accordance with this second aspect, it was surprisingly found that for long-term maintenance of viable three-dimensional tissue, there is need to apply rhythmic pulses of pressure (hydrostatic, mechanical or shear force) in order to obtain optimal growth. For example for growing of an articular cartilage tissue there is an advantage in maintaining the tissue under reparative cycles of loads and unloads of pressure in a rhythmic manner, simulating the natural growth conditions in the joint. The cellular mechanoreceptors seem to play a key role in this respect of cell growth.
The variables that can be manipulated in the system of the invention include stream flow velocity, amount (in atmospheres) of hydrostatic and/or mechanic pressure, rhythmic action periods (frequency of applications of pressure) and pausal intervals as well as change stream direction of the medium. By this second aspect, the present invention concerns a system for the maintenance of viable tissue comprising;
(i) a chamber for holding the tissue, the chamber""s atmosphere being kept at a relatively constant gas composition, said gas composition being suitable for maintenance of viable biological tissues;
(ii) a reservoir for holding tissue culture medium, said reservoir being in flow communication with the chamber;
(iii) a pump for circulating the medium between the chamber and the reservoir in a controlled manner; and
(iv) a pressure generator for producing rhythmic pulses of pressure on the tissue present in the chamber.
The system of the invention is suitable for any type of cells or tissues, but is especially suitable for the growth of a three-dimensional tissue, according to the definition above.
Basically, the system comprises a chamber for holding the tissue, the chamber""s atmosphere being kept at relatively constant gas and temperature composition which are suitable for maintenance of viable biological tissue, for example 5% to 10% CO2 in air at physiological temperature. This is usually achieved by placing the chamber within a larger CO2 incubator, capable of maintaining such an atmosphere, and ensuring that the atmosphere of the incubator in communication (as regards temperature and gas composition) with that of the chamber.
The system further comprises a reservoir for holding tissue culture medium which is in flow communication with the chamber. Preferably, the size of the reservoir is about 30 to 100 larger than that of the chamber for holding the tissue and is typically the size of 400-1000 ml. The medium in the reservoir of course contains the nutrients and various agents such as growth factors, etc. required for maintaining viability and growth of the tissue.
The system comprises a pump which circulates the growth medium between the chamber and the reservoir in a controlled manner. The pump may be a constant pump or a peristaltic pump utilizing computerized manipulated regimes, as will be explained hereinbelow. Typically, the velocity of medium flow is in the range of 300-600 ml/min.
The pressure generator may produce mechanical or hydrostatic pressure on the tissue and may be, for example, a compressor present in the chamber, which can periodically apply pressure on the tissue present in the chamber when streaming in one direction and the pressure is released when streaming in the other direction. The compressor should be under control of a control mechanism capable of controlling the timing (frequency, pausal, etc.) and level of compression, such as a clock or a computer mechanism.
The control mechanism would trigger the compressor, to compress the chamber thus applying rhythmic pressure on the liquid present therein, and consequently applying pressure on the tissue. In the case of a compressor, the pump""s activity may be constant so that the medium circulates between the chamber and the reservoir at a constant rate in order to improve gas exchange and nutrient availability to the tissue.
By another alternative, the pump that circulates the medium between the chamber and the reservoir is itself the pressure generator capable of producing rhythmic pulses of hydrostatic pressure on the tissue. In that case the pressure generator is the pump itself and no additional elements (such as a compressor) are required to produce the rhythmic pulses of pressure. The pump which is a perstaltic may have a built-in means for triggering rhythmic pulses. Alternatively, the pump may be connected to a control mechanism which triggers the duration, delays and frequencies of the pump such as a clock or a computer mechanism.
By alternating activities of such a pump, the medium can circulate in pulses between the medium reservoir and the chamber, thus creating rhythmic pressure pulses on the tissue. Preferably the direction of the medium flow should be changed (for example clock-wise and then counter-clock-wise) as changing the flow direction simulates best the joint""s conditions of loading and unloading. Typically change of direction should be every 1 to 3 min.
The rhythmic pulses should have a frequency of 5-300 per min., preferably 10-200 per min., most preferably 60 to 120 per min.
The hydrostatic pressure should be between 0.5 and 30 atm., preferably 1 to 10 atm, most preferably 2 to 3 atm.
The present invention concerns a method for maintaining viable tissue, cells or extracts from three-dimensional tissue, comprising placing these tissues in the chamber of the above system with any of the above parameters of pressure, frequency and change of flow direction. Examples of tissue are as defined above in connection with the scaffold.
The present invention further provides a method for maintaining viable cells or tissue extracts from three-dimensional tissue comprising growing a prosthesis composed of the scaffold of the invention impregnated with cells in the system of the invention with conditions specified above (i.e. the parameters specified above). A preferable example is a method for growing epiphyseal tissue.